BioVision rev.

05/16
For research use only

DNA Damage Quantification Colorimetric Kit

(Catalog #K253-25; 25 assays; Store kit at 4°C) B. ARP Labeling:

I. Introduction: 1) Mix 5 µl of 0.1 µg/µl purified sample DNA solution with 5 µl ARP Solution at the bottom of a
Apurinic/apyrimidinic (AP) sites are one of the major types of DNA lesions formed during microcentrifuge tube and incubate at 37°C for 1 hour to tag the DNA AP site.
the course of base excision and repair of oxidized, deaminated or alkylated bases. It has 2) Add 88 µl TE and 2 µl Glycogen to the reaction solution, mix well.
been estimated that about 2x105 base lesions are generated per cell per day. The level of 3) Add 0.3 ml of pure ethanol (not provided) mix well and keep at –20°C for 10 min.
AP sites in cells can be a good indicator of DNA lesion and repair against chemical Centrifuge with microcentrifuge at the top speed for 10 min to precipitate the AP-site tagged
damage and cell aging. The DNA Damage Quantification Kit utilizes the ARP (Aldehyde DNA.
Reactive Probe) reagent that reacts specifically with an aldehyde group which is the open
4) Wash the pellet three times with 0.5 ml 70 % ethanol. Quick spin to remove the trace
ring form of the AP sites. After treating DNA containing AP sites with ARP reagents, AP
amount of ethanol. Air dry the pellet for 5 min. The Biotin-tagged genomic DNA pellet can
sites are tagged with biotin residues, which can be quantified using avidin-biotin assay
be used immediately or store at -20°C. The tagged DNA sample is stable for at least one
followed by a colorimetric detection. The kit provides the necessary reagents for
year.
convenient determination of abasic sites in purified DNA sample in 96-well plate format.
II. Kit Contents: C. Determination of the number of abasic sites in DNA
1) Dilute the 40 ARP-DNA Standard (40 ARP sites per 105 bp) with 0 APR-DNA Standard to
Component K253-25 Color Code Part generate 200 µl each of the 0, 8, 16, 24, 32, 40 ARP-DNA solutions in microcentrifuge
tubes (see below table).
25 assays Cap Color Number
ARP Number 0 8 16 24 32 40
ARP Solution (10 mM, in DMSO) 0.125 ml Red K253-25-1
40 ARP-DNA Standard (µl) 0 40 80 120 160 200
TE Buffer 30 ml NM K253-25-2
Glycogen Solution (10 µg/µl) 0.1 ml Blue K253-25-3 0 ARP-DNA Standard (µl) 200 160 120 80 40 0
0 ARP-DNA Standard (0.5 µg/ml) 0.6 ml Clear K253-25-4 2) Dissolve the Biotin-tagged DNA samples prepared in B with 1 ml of TE buffer (0.5 µg/ml).
40 ARP-DNA Standard (0.5 µg/ml) 0.6 ml Yellow K253-25-5
3) Add 60 µl each of the above ARP-DNA Standards and ARP-labeled DNA samples into
DNA Binding Solution 10 ml NM K253-25-6 each well. For more accurate measurement, use three wells per sample.
HRP-Streptavidin 0.1 ml Green K253-25-7
4) Add 100 µl of the DNA Binding Solution to the standards and samples, keep the plate at
10X Wash Buffer 30 ml WM K253-25-8 room temperature overnight to allow the tagged-DNA bind on the plate surface. Keep the
HRP Developer 10 ml Brown/NM K253-25-9 wells sealed.
96-well Microplate (8 x 12 strips) 1 plate Clear K253-25-10 Prepare Solutions before use:
Washing Buffer: Dilute the 10X Wash Buffer to 1X Buffer with ddH2O (total volume 300
III. DNA Damage Quantification Protocol:
ml). Store this 1X Wash Buffer at room temperature.
A. Purification of Genomic DNA:
HRP-Streptavidin Solution: Just before use, prepare 1:100 diluted working solution by
Several different methods and products are available for isolating genomic DNA. Among diluting the 100 µl of HRP-Streptavidin with 9.9 ml 1X Wash Buffer.
all the methods, the guanidine/detergent lysis method is simple, and it gives highly purified
5) Discard the DNA Binding Solution in the wells, and wash the well with 250 µl Wash Buffer 5
genomic DNA for the ARP-based abasic sites detection. During the purification process,
times.
avoid heating of the DNA solution. Determine the concentration and purity of the purified
genomic DNA using the spectrophotometer*. Dissolve the genomic DNA in TE at 6) Add 100 µl diluted HRP-Streptavidin solution to each well, and shake the plate for 1 hr at
concentration of 0.1 µg/µl. It is important for an accurate assay that the DNA concentration room temperature.
is adjusted exactly to 0.1 µg/µl. 7) Discard the solution in the wells, and wash the wells with 250 µl Wash Buffer 5 times.
1.0 OD260 nm = 50 µg/ml for genomic DNA. The ratio of OD260 nm/OD280 nm of highly purified 8) Add 100 µl of HRP Developer to each well, and incubate at 37°C for 1 hour.
DNA solution is 1.7 or higher. Protein contamination in the sample solution may cause a 9) Measure the OD 650 nm (within 1 hour after the incubation is ended), or add 100 µl of 1 M
positive error. Sulfuric acid (or 6 M HCl) to stop the reaction. Mix well and measure OD 450 nm.*
Note: Genomic DNA Isolation Kit is also available from BioVision (Cat.# K281-50). For a *Note: The value from OD 450 nm will be roughly double of that from OD 650 nm.
positive control, cells may be treated using 10 mM H2O2 for 1 hour at 37°C to induce AP 10) Prepare the calibration curve using the data obtained with standard ARP-DNA solutions.
site formation. Apply your sample DNA readings to the Standard Curve. The ARP numbers are the basic
sites per 105 bp in the genomic DNA samples. Compare the numbers of AP sites in treated
samples vs control samples to determine the level of DNA Damage.
FOR RESEARCH USE ONLY! Not to be used on humans.

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